PROJECT SUMMARY/ABSTRACT Cholangiocarcinoma (CC) is among the deadliest cancers worldwide, with a five-year survival rate of ~30% in intrahepatic cholangiocarcinoma (ICC) and ~50% in extrahepatic cholangiocarcinoma (ECC). It is a rare cancer, affecting ~8,500 (5,500 ICC, 3000 ECC) patients in the U.S. each year. Often cancers develop from an imbalance between signals that regulate cellular growth and differentiation. While most advances in cancer therapy have been made by inhibition of cell proliferation, the re-establishment of cell differentiation has resulted in the cure of certain forms of leukemia. In this proposal, we will focus on the interaction between hepatocyte nuclear factor 4 alpha (HNF4?), the master regulator of hepatic differentiation, and the Wnt/?- catenin signaling pathway, which enhances cell proliferation. We will use zebrafish for our studies, which has advantages as a model organism because of in vivo imaging, time resolution, and chemical screening methods not available or feasible in rodent models. Importantly, zebrafish display disease morphology and pathophysiology that is similar to mammals. We hypothesize that an HNF4?/?-catenin interaction is important in hepatobiliary development playing an important role cell fate during both development and cholangiocarcinoma formation. Our first aim is to characterize and define the importance of HNF4?/?-catenin interaction in regard to hepatobiliary development. We have recently generated novel HNF4? knockout zebrafish and are in the process of generating a heat shock-inducible HNF4? over-expression zebrafish. These models will allow us to not only define the role of HNF4? in hepatobiliary development, but, in combination with genetic and pharmacologic models of Wnt/?-catenin modulation, will allow us to characterize an interaction between these two pathways. We will use these mutants and and chemical modulators to characterize liver growth, morphology, and physiology during hepatobiliary development and assess perturbations in normal hepatic and biliary cell fate. Our second aim is to determine a role for the HNF4?/?- catenin interaction in cholangiocarcinoma formation. We will first characterize tumor incidence, progression, and mortality in zebrafish with mutations resulting in dysregulation of the HNF4?/?-catenin pathway. Further, we will utilize both genetic and pharmacologic means to re-establish a normal HNF4?/?-catenin interaction and observe changes in tumor burden. These studies will not only reveal novel mechanisms of cholangiocarcinoma pathogenesis, but also will allow us to develop new targets for therapeutic development for this devastating disease.